The research proposed in this application is designed to evaluate the contributions made by mRNA-rRNA interactions, initiator codon sequence, and mRNA secondary structure in determining the specificity of ribosome recognition and the overall efficiency of a translation initiation site. The wild type initiator for the lactose repressor protein of E.coli and internal reinitiation sites activated by nonsense mutations early in the I gene encoding the repressor will be examined in detail. My recent determination of the 5' terminal 214 nucleotides of the repressor mRNA (I mRNA) sequence has identified those regions which function as the wild type initiator and at least three internal restart sites, but in addition, has raised a number of questions about the molecular features which are significant in the in vivo functioning of this messenger. Experiments using the combined techniques of mRNA and DNA sequencing, in vitro ribosome binding, and N-terminal amino acid sequencing are proposed to analyze I gene mutants specifically affected in initiation, with the aim of 1) dissecting the functional elements of the wild type initiator and of 2) determining which I mRNA sequence and structural features underlie the apparent specificity of reinitiation site selection. The ease of fine structure genetic analysis, nucleic acid sequence determination, and identification of in vivo translational start sites provided by this experimental system is expected to result in substantial contributions by this study to our understanding of the molecular interactions which underlie and regulate this step of gene expression.